Gonadotropin-releasing Hormone Antisera Research Articles

Development and Differentiation of Gonadotropin Hormone-Releasing Hormone Neuronal Systems and Testes in the Japanese Eel (Anguilla japonica)

In this study we investigated the relationship between the development of the olfactory, preoptic, and midbrain gonadotropin-releasing hormone (GnRH) neuronal systems and testicular differentiation in eels (Anguilla japonica) from embryonic stages through adulthood (5.4–50 cm body length). GnRH-synthesizing neuronal populations were first observed in the youngest fish (∼5.0 cm) at the rostrobasal and caudalmost olfactory bulbs immunoreactive to a “promiscuous” (nonspecific) GnRH antiserum (635.5), and in the preoptic area and midbrain tegmentum immunoreactive to chicken GnRH II antiserum. The eel brains lacked salmon and seabream GnRH immunoreactivity. The evidence from our study suggests that the olfactory, preoptic, and midbrain GnRH populations have origins independent from those of proliferative periventricular zones within the brain. However, the olfactory GnRH neurons could have migrated out of the olfactory placodes during ages earlier than those observed in this study. Although all three GnRH neuronal populations contribute to pituitary innervation to some degree, the preoptic GnRH innervation was pronounced in the pituitary when primordial germ cells (animals ∼5.0 cm) differentiated into male germ cells (animals 14–16 cm) and, therefore, an association can be assumed between preoptic GnRH expression and testicular differentiation in the Japanese eel.

Distribution of gonadotropin-releasing hormone immunoreactive systems in the brain of the Senegalese sole, Solea senegalensis.

The present paper reports the immunohistochemical distribution of the gonadotropin-releasing hormone (GnRH) structures in the brain of the Senegalese sole, Solea senegalensis. In this study, we have used two antibodies against the salmon GnRH and chicken GnRH-II forms and the streptavidin-biotin-peroxidase complex method. Immunoreactive cell bodies are observed at the junction between the olfactory bulbs and the telencephalon (terminal nerve ganglion cells), in the ventral telencephalon, in the preoptic parvocellular nucleus, and in the synencephalic nucleus of the medial longitudinal fasciculus. GnRH-immunoreactive fibres were found extensively throughout the brain, located in the telencephalon, preoptic area, hypothalamus, hypophysis, optic tectum, midbrain and rhombencephalon. The antisera used in this study against the two GnRH forms exhibited cross-reactivity on the same cell masses and did not allow cell populations expressing different GnRH forms to be discriminated clearly. However, anti-salmon GnRH immunostained the GnRH cells and fibres of the forebrain much more intensely, whereas the anti-chicken GnRH antiserum shows a higher immunoreactivity on synencephalic cells of the medial longitudinal fasciculus.

Effects of bovine follicular fluid and passive immunization against gonadotropin-releasing hormone (GnRH) on messenger ribonucleic acid for GnRH receptor and gonadotropin subunits in ovariectomized ewes.

In cultured ovine pituitary cells, inhibin increases concentrations of mRNA encoding GnRH receptor and numbers of GnRH receptors. The objective of this study was to test the hypothesis that inhibin increases concentrations of ovine GnRH receptor mRNA in vivo. Ovariectomized ewes were used to eliminate effects of endogenous ovarian hormones, and passive immunization against GnRH was employed to avoid possible confounding influences of GnRH on GnRH receptor gene expression. Two groups of ewes (n = 5/group) were treated with 50 ml GnRH antiserum on Days 0 and 3 of the experiment. One group of immunized ewes received 10 ml charcoal-extracted bovine follicular fluid (bFF) as a source of inhibin every 8 h for 48 h on Days 4-6 of the experiment. A third group of ewes was not passively immunized and was treated only with bFF, and control ewes received no treatments. Anterior pituitary glands were collected from all ewes on Day 6. Passive immunization against GnRH, alone or in combination with treatment with bFF, decreased mean concentrations of LH (p < 0.01) and LH pulse amplitude (p < 0.001). In ewes treated only with GnRH antiserum, number of LH pulses was also reduced (p < 0.03). Circulating concentrations of FSH tended to be lower (p = 0.06) in passively immunized ewes compared to controls. Treatment with bFF, alone or in combination with GnRH antiserum, reduced circulating concentrations of FSH (p < 0.02) and amounts of FSHbeta subunit mRNA (p < 0.001) to less than 30% and 10% of control values, respectively. Despite effects of bFF on concentrations of FSHbeta mRNA and secretion of FSH, concentrations of GnRH receptor mRNA were similar among controls, ewes treated with bFF alone, and passively immunized ewes treated with bFF. Passive immunization against GnRH did not affect concentrations of GnRH receptor mRNA but resulted in a reduction (p < 0.05) in amount of LHbeta mRNA. Treatment with bFF did not affect amounts of either alpha subunit or LHbeta subunit mRNA except when combined with treatment with antiserum, when amounts of both alpha and LHbeta subunit mRNA were reduced (p < 0.05). These results do not support the hypothesis that inhibin increases concentrations of GnRH receptor mRNA in the ewe, and they provide evidence that inhibin is not an acute regulator of ovine GnRH receptor gene expression in vivo.

Mast cells with gonadotropin-releasing hormone-like immunoreactivity in the brain of doves.

Using an antiserum (LR-1) raised against mammalian gonadotropin-releasing hormone (GnRH), we previously identified a nonneuronal cell that was more numerous in the medial habenula (MH) of courting ring doves than in individuals housed in visual isolation. The current studies suggest that they are mast cells. Both acidic toluidine blue and toluidine blue dissolved in water/butanediol revealed metachromatic cells with a distribution and morphology similar to that obtained by immunostaining with the GnRH antiserum in the MH. Some cells had granules reactive to safranin in the presence of alcian blue, indicative of a highly sulfated proteoglycan of the heparin family. Immunocytochemical studies demonstrated that all MH cells containing GnRH-like immunoreactivity contained histamine, another mast cell marker. The GnRH-immunoreactive cells had a unilobular, ovoid nucleus. Secretory granules within the cells were electron dense and displayed a variety of internal structures. Fine filamentous processes appeared evenly distributed on the cell surface whether cells were located on the pial surface or within the brain parenchyma. All of these features are characteristic of mast cells. To test whether the epitope recognized by the GnRH antiserum was produced by the mast cells or endocytosed from the cerebrospinal fluid, an iodinated GnRH analog was injected intracerebroventricularly at the initiation of courtship. Radioautography revealed no radioactive cells in the brain, indicating that the GnRH antibody recognized a molecule synthesized by the nonneuronal cells rather than internalized by a receptor-mediated mechanism. These observations suggest an interaction between a component of the immune network and specific regions of the central nervous system.

Passive immunization of the pig against gonadotropin releasing hormone during the follicular phase of the estrous cycle

Three experiments were conducted to determine the effects of passively immunizing pigs against gonadotropin releasing hormone (GnRH) during the follicular phase of the estrous cycle. In Experiment 1, sows were given GnRH antibodies at weaning and they lacked estrogen secretion during the five days immediately after weaning and had delayed returns to estrus. In Experiment 2, gilts passively immunized against GnRH on Day 16 or 17 of the estrous cycle (Day 0 = first day of estrus) had lower (P<0.03) concentrations of estradiol-17β than control gilts, and they did not exhibited estrus at the expected time (Days 18 to 22). When observed three weeks after passive immunization, control gilts had corpora lutea present on their ovaries, whereas GnRH-immunized gilts had follicles and no corpora lutea. The amount of GnRH antiserum given did not alter (P<0.05) serum concentrations of LH or pulsatile release of LH in sows and gilts. In Experiment 3, prepuberal gilts were given 1,000 IU PMSG at 0 h and GnRH antiserum at 72 and 120 h. This treatment lowered the preovulatory surge of LH and FSH, but it did not alter serum estradiol-17β concentrations, the proportion of pigs exhibiting estrus, or the ovulation rate. These results indicate that passive immunization of pigs against GnRH before initiation of or during the early part of the follicular phase of the estrous cycle retards follicular development, whereas administration of GnRH antibodies during the latter stages of follicular development does not have an affect. Since the concentration of antibodies was not high enough to alter basal or pulsatile LH secretion, the mechanism of action of the GnRH antiserum may involve a direct ovarian action.

Intratesticular Control Of Steroidogenesis

Men are men because of testosterone, and the effects of this hormone are evident in virtually every organ or tissue in a normal adult man. Because testosterone is a classical endocrine hormone with effects throughout the body, many endocrinologists will have failed to realize that, in terms of its effects on fertility, the primary role of testosterone is not endocrine but paracrine, as it is the local action of testosterone on spermatogenesis which determines fertility. When looked at from this perspective it is logical to question the traditional endocrine teaching which states that testosterone secretion is controlled solely by pituitary luteinizing hormone (LH). Certainly, the levels of testosterone and LH in peripheral blood show a close relationship, but it is not the level of testosterone in peripheral blood which determines fertility. If the primary role of testosterone is within the testis, then it is not unreasonable to expect that local mechanisms may exist to regulate the intratesticular levels of this steroid. The object of this review is to examinecritically the evidence for such mechanisms, their possible nature and their physiological and clinical significance (if any). Before reviewing the data on intratesticular control of steroidogenesis it is essential to place it in perspective. To do this, three principal questions need to be answered: (I) Why might intratesticular mechanisms for local control of testosterone production be required? (2) What is the role (or roles) of testosterone within the testis? A proper understanding of how testosterone controls spermatogenesis is essential if the local regulation of testosterone production is to be evaluated from a physiological perspective. (3) What are the constraints or problems relating to studies on the intratesticular control of steroidogenesis? It is essential to understand the potential limitations of studies on this subject so that a reasoned interpretation of the results can be made. Most relevant data is derived from studies in uitro, and the relationship of such data to the situation in uivo is often difficult to establish.

Testing of Arg-8-gonadotropin-releasing hormone-directed antisera by immunological and immunocytochemical methods for use in comparative studies

Three polyclonal antisera raised in rabbits against the mammalian molecular form of gonadotropin-releasing hormone (GnRH) were tested in enzyme-linked immunosorbent assays for crossreactivity with naturally occurring GnRHs and with GnRH analogues. Antisera were then tested immunocytochemically in order (i) to identify amino acids essential for the binding of each antiserum, and (ii) to evaluate the specificity of the immunocytochemical reaction in brain sections from various species of cyclostomes, amphibians, reptiles, and birds. Antiserum GnRH 80/1, recognizing mainly a discontinuous determinant including the NH2- and COOH-termini, crossreacts with GnRHs the molecular bending of which enables the spatial approach of both terminal amino acid residues. Antiserum GnRH 80/2, by requiring the COOH-terminus for binding and not tolerating substitutions by aromatic amino acids in the middle region of the molecule, recognizes chicken I GnRH, however, not the salmon form. The use of this antiserum is appropriate in species synthesizing the mammalian and/or the chicken I form of GnRH. GnRH antiserum 81/1 is specific mostly for mammalian GnRH. The results obtained by ELISAs are confirmed by immunocytochemical studies. A comparison between the results obtained in ELISA and in immunocytochemistry involving mammalian-, chicken I-, chicken II-, salmon-, and lamprey-directed GnRH antisera resulted in the following conclusions: (1) An antiserum recognizing the discontinuous antigen determinant including both NH2- and COOH-termini may be reactive in most vertebrate brain sections thus being appropriate for phylogenetically directed immunocytochemical studies. (2) Moreover, this discontinuous determinant seems to be immunocytochemically reactive in all parts of the neurons in the GnRH system, whereas, in some species, determinants located in the middle region of the molecule(s) tend to become reactive only during the axonal transport. (3) A crossreaction between tissue-bound antigen and antibodies recognizing the above cited discontinuous determinant indicates an appropriate bending of the molecule even in case of severe molecular differences, e.g., in lamprey form of GnRH. (4) It follows that in phylogenetic studies, an immunologically well characterized antiserum can be substituted for a species-directed antiserum.

Effects of Ovariectomy on GnRH mRNA, proGnRH and GnRH Levels in the Preoptic Hypothalamus of the Female Rat

Experiments were carried out to investigate the effects of ovariectomy on gonadotropin-releasing hormone (GnRH) messenger RNA (mRNA), proGnRH and GnRH peptide levels in the hypothalamus of female rats. Intact proestrous female rats and female rats, which had been ovariectomized for 2 weeks, were sacrificed at 9.00 h and the preoptic area (POA) and basal hypothalamus (BH) were dissected out and frozen on dry ice. One group of tissues from proestrous control and ovariectomized females were extracted in acetic acid, centrifuged at 13,000 g and the supernatant purified on a C18 column. The purified extract was then radioimmunoassayed for proGnRH, using a specific antiserum to rat proGnRH (ARK-2), and for GnRH using the E1-14 antiserum. Total cellular RNA was isolated from another group of tissues and prepared as Northern blots. Hybridization with 32P-labeled GnRH cRNA was used to detect GnRH mRNA. A third group of proestrous and ovariectomized female rats were perfused, and 50 microns vibratome sections were cut. These were immunostained with proGnRH or GnRH antiserum, followed by in situ hybridization with 35S-labeled GnRH cRNA to detect GnRH mRNA. Based on the histochemical staining, mRNA was colocalized to the cell soma of neurons containing proGnRH and GnRH throughout the POA and BH. Based on the radioimmunoassay, proGnRH levels were 2 times higher in the POA versus the BH, but GnRH levels were 6-7 times higher in the BH. Ovariectomy significantly decreased proGnRH levels in both the POA and BH, while GnRH decreased in the BH. In contrast, quantitative Northern blot analysis demonstrated that ovariectomy had no effect on mRNA levels in the POA and BH. These data indicate that the effects of ovariectomy on proGnRH and GnRH levels are a result of altered translation, posttranslational processing and/or secretion of GnRH.

Effect of passive immunization to gonadotropin-releasing hormone (GnRH) using GnRH antiserum on the mitotic activity of gonadotrophs in castrated male rats.

In order to reveal the mechanism of elevation in the mitotic activity of gonadotrophs in the pituitary gland of castrated rats, passive immunization to GnRH designed to block the activity of GnRH in castrates was performed, and changes in the mitotic activity of pituitary gonadotrophs and mammotrophs were studied. The increased serum levels of gonadotropins and their subunits in castrates were dramatically suppressed by the administration of rabbit anti-GnRH serum (RAGnRH). However, this treatment had no effect on the serum levels of PRL, suggesting that the passive immunization to GnRH used in this study was only effective in blocking the hormonal activity of GnRH to the gonadotropin secretion. Mitosis of the gonadotrophs in normal rat anterior pituitary was rarely observed, but it was dramatically increased by castration. This elevated mitotic activity of gonadotrophs in the castrated rats was significantly suppressed by the administration of RAGnRH. On the other hand, mitotic activity of mammotrophs was decreased by castration. This diminution in the mitotic activity of mammotrophs was not changed by administration of RAGnRH. These results showed that GnRH is an important factor for stimulation of gonadotroph cell proliferation in castrated rats.

Differential distribution of two molecular forms of gonadotropin-releasing hormone in discrete brain areas of goldfish ( Carassius auratus)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were identified in the extracts of various brain areas, spinal cord and pituitary in female and male goldfish and had chromatographic and immunological properties similar to [His 5,Trp 7,Tyr 8]-GnRH (cGnRH-II) and [Trp 7,Leu 8]-GnRH (sGnRH). Radioimmunoassay using different GnRH antisera after high pressure liquid chromatography did not reveal significant peaks of mammalian GnRH, [Gln 8]-GnRH and [Tyr 3,Leu 5,Glu 6,Trp 7,Lys 8]-GnRH in the brain extracts. The proportion of cGnRH-II-like immunoactivity to sGnRH-like immunoactivity was higher in the caudal brain areas compared to the rostral areas. The differential distribution of two GnRH forms suggest that the different GnRH forms may have different physiological functions.

Gonadotropin-releasing hormone from brains of reptiles: Turtles ( Pseudemys scripta) and snakes ( Thamnophis sirtalis parietalis)

Gonadotropin-releasing hormone (GnRH)-like peptides were present in whole brain extracts of turtle ( Pseudemys scripta) and snake ( Thamnophis sirtalis parietalis) with higher content and concentration in the turtle brain. The peptides were identified by cross-reactivity profiles with four GnRH antisera and by retention times on reverse-phase high-pressure liquid chromatography (HPLC) compared with synthetic GnRH standards. Turtle brain extracts contained two HPLC peaks that cross-reacted with GnRH antisera; these peaks eluted from the HPLC in the same positions as chicken I and II GnRH. Snake brain extracts contained only one major HPLC peak (and two minor peaks in some brains) that cross-reacted with anti-GnRH sera; the major peak eluted with the same retention time as chicken I GnRH. Mammalian, salmon, and lamprey GnRH-like peptides were not detected. In extracts from both turtle and snake brains, the cross-reactivity profile of the HPLC peaks compared with those of synthetic chicken I and II GnRH showed a similar order of sensitivity with four antisera. It is likely that chicken I and II GnRH-like peptides were present in ancestral reptiles prior to the evolution of the three living reptilian subclasses of Anapsida (turtle), Lepidosauria (snake and lizard), and Archosauria (alligator). This assertion is based on the present demonstration and work by others showing that chicken I and II GnRH-like peptides are in turtle and alligator, chicken I is in snake, and chicken II is in lizard.

The role of endogenous gonadotropin-releasing hormone in the control of luteinizing hormone and testosterone secretion in the juvenile male monkey, Macaca fascicularis.

To determine what changes occur in the activity of gonadotropin-releasing hormone (GnRH) neurons during pubertal development in primate species we tested the hypotheses that there are morphologic differences between GnRH-containing neurons in juvenile versus adult monkeys, and the low activity of the reproductive axis is governed by hypothalamic GnRH release in monkeys prior to puberty. We removed the brains from 5 juvenile and 5 adult male monkeys (Macaca fascicularis) and blocked, sectioned, and prepared each hypothalamus for light microscopic immunocytochemistry for GnRH-containing cells. The distribution and number of GnRH-containing neurons were similar in adult and juvenile brains; however, GnRH-containing perikarya in adult brains were significantly larger in total cross-sectional area (200 +/- 12 vs. 169 +/- 8 micron 2, P less than 0.05) and in cross-sectional area of the cytoplasm (139 +/- 2 vs. 88 +/- 6 micron 2, P less than 0.05) than in juvenile brains. In another group of 10 juvenile male macaques, we administered an antiserum to GnRH (Fraser #94; 2 ml/kg, i.v.) and monitored the effects on plasma luteinizing hormone (LH) and testosterone concentrations. The percentage of plasma samples with detectable LH levels decreased significantly (from 26.67 +/- 8.3% to 5.3 +/- 3.4%, P less than 0.05) after GnRH antiserum administration; however, plasma testosterone concentrations (0.08 +/- 0.02 ng/ml) remained unchanged. We conclude that during pubertal maturation in primate species there is increased synthesis and release of GnRH from a population of GnRH neurons that are active prior to puberty.

Correlation of LH, FSH and GnRH release during various brain-pituitary coincubations.

Hemipituitaries and various brain fragments from female albino rats were coincubated in the presence or absence of veratrine (a depolarizing agent). The release of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH) was measured by radioimmunoassay. In the absence of veratrine, none of the brain fragments affected LH and FSH release. In the presence of veratrine, large increases in LH and FSH release were caused by medial basal hypothalamic (MBH) fragments, slight increases were caused by suprachiasmatic:preoptic area fragments, and cerebral cortex fragments had no effect. LH and FSH release caused by brain fragments was significantly correlated with GnRH release and the GnRH concentrations attained during coincubations could account for all increases in LH and FSH release observed. Also, GnRH antiserum completely blocked increases in LH and FSH release during MBH-hemipituitary coincubations. The data indicate that the rat brain is unlikely to contain neurohormones other than GnRH capable of directly stimulating LH and FSH release in vivo.

Neutralization of gonadotropin-releasing hormone in neonatal rats with permanent impairment of the hypothalamic-pituitary-testicular axis.

Males rats were passively immunized at 5 days of age with a single 0.25 ml i.p. injection of gonadotropin-releasing hormone (GnRH) antiserum. Control animals were given an equal volume of normal rabbit serum (NRS). Serial blood determinations of gonadotropins, testosterone and dihydrotestosterone (DHT) were obtained at intervals ranging from early in life through adult life. Gonadotropin secretion was reduced (P less than 0.025) up to 35 days of age. Androgen secretion (testosterone) was reduced (P less than 0.05) at 10 and 33 days of age. When hCG was given to 54-day-old (young adult), and 100-day-old and 15-month-old animals, testosterone concentrations were similar in both experimental and control groups 1 h after hCG stimulation. As adults, basal gonadotropins were the same in both groups; however, after GnRH stimulation, the GnRH antiserum-treated groups showed an increased gonadotropin response when compared to the NRS control group. In order to determine whether there was an alteration in steroid feedback, other animals were castrated at adult age (approximately 100 days old), and exogenous testosterone was given in increasing increments. However, serum gonadotropins decreased similarly in treated and control groups. These data indicate that a single injection of GnRH antiserum early in life decreased gonadotropin secretion temporarily during prepubertal sexual development and caused a permanent alteration in hypothalamic-pituitary-testicular function.